Atrial natriuretic peptide (hereinafter referred to as “ANP”), brain natriuretic peptide (hereinafter referred to as “BNP”), C-type natriuretic peptide (hereinafter referred to as “CNP”) and Dendroaspis natriuretic peptide (hereinafter referred to as “DNP”) are each members of a family of hormones known as “natriuretic peptides”. ANP and BNP share a wide spectrum of biological properties and belong to the cardiac natriuretic system. Both ANP and BNP are of myocardial cell origin while CNP is of endothelial cell origin. DNP was isolated from the venom of the green mamba snake and possesses structural similarity to ANP, BNP and CNP.
BNP received its name because it was first isolated from porcine brain, thus “BNP” stood for “brain natriuretic peptide”. However, because BNP belongs to the cardiac natriuretic system, “brain” has been changed to “B-type”. Therefore, “BNP” now refers to “B-type natriuretic peptide”.
ANP is secreted by the heart in the atria. BNP is secreted by the heart through the coronary sinus, predominantly from the cardiac ventricles. BNP is secreted as a 108 amino acid polypeptide precursor (See Valli et al., J. Lab. Clin. Med., 134(5):437–444 (November 1999)). The mature form of BNP is made up of 32 amino acids with a 17 amino acid ring closed by a disulfide bond between two cysteine residues, an amino-terminal tail of 9 amino acids, and a carboxyl-terminal tail of 6 amino acids. ANP and CNP also have a 17 amino acid ring closed by a disulfide bond between two cysteine residues. Eleven of the seventeen amino acids in the ring are conserved between the three molecules. In addition to the 17 amino acid ring structure, ANP has an amino-terminal tail of 6 amino acids and a carboxy-terminal tail of 5 amino acids. ANP is produced as a 126 amino acid pro-ANP form that is the major storage form of ANP. After proteolytic cleavage between amino acids 98 and 99, the mature 28 amino acid peptide ANP is found in coronary sinus plasma (See Yandle, J. Internal Med., 235:561–576 (1994)).
CNP is found in the brain and cerebral spinal fluid and is the most prevalent of the three peptides in the central nervous system. Little if any CNP is present in the heart. Pro-CNP is a 103 amino acid peptide that is processed into either CNP-53 (amino acids 51 to 103) or CNP-22 (amino acids 82 to 103) that are the active peptides. In addition the 17 amino acid ring structure, CNP-22 has an amino-terminal tail of 5 amino acids and contains no carboxy-terminal tail. CNP-53 is identical to CNP-22 except for a 31 amino acid extension at the amino terminal end.
As mentioned previously, DNP was isolated from the venom of the green mamba snake. The mature form of DNP is made up of 38 amino acids. DNP-like immunoreactivity (DNP-LI) has been reported in human plasma and the plasma concentration of DNP-LI has been found to be elevated in patients with congestive heart failure (See, Cataliotti, et al., Mayo Clin. Proc., 76:111–1119 (2001)). Additionally, it is also known that the infusion of synthetic DNP results in marked natriuresis and diuresis in association with increased plasma and urinary cyclic guanosine monophosphate. Id.
The measurement of BNP in human plasma in the general population has been found to reflect cardiac diseases, such as congestive heart failure, ischemic heart diseases, atrial fibrillation and renal dysfunction. In fact, elevated levels of BNP in human plasma has been reported in heart disease, following acute myocardial infarction and during symptomless or subclinical ventricular dysfunction (See Mukoyama et al., J. Clin. Invest., 87:11402–11412 (1991), Motwani et al., Lancet, 341:1109–1113 (1993), Yoshibayashi et al., New Eng. J. Med., 327:434 (1992)). Increased circulating levels of ANP are seen in congestive heart failure, chronic renal failure and in severe hypertension. The presence of CNP in human plasma remains controversial with reports of its absence or presence as CNP-22 (See Yandle, J. Internal Med., 235:561–576 (1994)).
A ligand binding assay is an analytical technique for measuring concentrations of substances commonly referred to as ligands that react selectively with specific binding proteins. Immunoassays that measure the concentrations of antigens that react selectively with specific antibodies are an example of a class of ligand binding assays.
Ligand binding assays, such as immunoassays, for measuring human natriuretic peptides in plasma are well-known in the art and are commercially available. These immunoassays require the use of at least one or two specific antibodies as well as at least one calibrator and, ideally, at least one control. Calibrators are used in ligand binding assays to calibrate instruments prior to calculating the sample result. The calibrators and controls used in such assays are typically made from human synthetic natriuretic peptides. Human synthetic natriuretic peptides are commercially available from a variety of sources. For example, human synthetic BNP is commercially available from Peptide Institute (Osaka, Japan), American Peptide Company, Inc. (Sunnyvale, Calif.), Synpep Corporation (Dublin, Calif.) and Phoenix Pharmaceuticals, Inc. (Belmont, Calif.).
One of the problems with both natural and synthetic human natriuretic peptides is that they are unstable in plasma and serum. Specifically, enzymes, such as proteases, cleave these peptides. For example, proteases cleave BNP (natural and synthetic) at various locations along its amino acid chain. For example, protease cleavage is known to occur at the amino terminus of BNP between amino acids 2–3 (Shimizu et al., Clinica Chimica Acta, 316:129–135 (2002)) and at its carboxy terminus between amino acids 30–32. Moreover, endopeptidase cleavage of BNP is also known in the art (Davidson and Struthers, J. Hypertension, 12:329–336 (1994)). Such cleavage is problematic because in order for calibrators and controls to function properly in an assay, a human natriuretic peptide containing immunoreactive or specific ligand binding sites must be present at the intended concentration during the assay.
Thereupon, as a result of this instability, the calibrators and controls used in such assays are sold either in lyophilized form (such as in the Shionoria assay available from Shionogi & Co., Ltd., Osaka, Japan) or frozen (such as in the Triage® assay available from Biosite, Inc., San Diego, Calif.). Calibrators and controls in lyophilized form must be reconstituted in a solvent prior to use in an assay. Once reconstituted, these calibrators and controls must be used within a specific time because they are very unstable. Calibrators and controls that are frozen (usually at temperatures of about −20° C. or colder) remain frozen until thawed for use in the assay. These calibrators and controls are thawed at room temperature and then vortexed or inverted to make the calibrators and controls homogenous prior to testing. Frozen calibrators and controls cannot be re-frozen and can only be used once (i.e. are single-use) and then discarded.
Thereupon, there is a need in the art for new calibrators and controls for use in human natriuretic ligand binding assays that are stable for extended periods of time and are easy and quick to use and do not need to be reconstituted or thawed prior to use in such assays.